Determination if thiamine in baby cereals PDF

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THE DETERMINATION OF THIAMINE IN VARIOUS BABY CEREALS BY USING SPECTROFLUOROMETRIC MEANS ABSTRACT Thiamine or vitamin B1 is a vitamin complex that is involved in several biological activities such as the metabolism of macromolecules e.g. carbohydrates, lipids and proteins. The aim of this experiment was to determine the thiamine concentration in baby cereals by means of a spectrofluorometer. A spectroflourometer was used to measure the fluorescence of thiamine at a basic pH as thiochrome with an excitation wavelength of 365nm and an emission wavelength of 435nm. The thiamine content of the cereals were obtained by the interpolation of their fluorescence reading from a standard curve constructed from samples of thiamine-HCl of known stock concentration (2mg/100ml). The purity samples yielded the lowest thiamine concentration when compared to the thiamine concentration on the cereal box. A higher thiamine yield was obtained from the proNutro samples and the cerelac samples had the highest amount of thiamine when compared to their true thiamine content on the box. INTRODUCTION Thiamine or vitamin B1 is a vitamin complex that is involved in several biological activities such as the metabolism of macromolecules e.g. carbohydrates, lipids and proteins (Elisa et al, 2006). It is obtained from food products that are rich in carbohydrates or food sources associated with a high carbohydrate metabolism (Coultate, 2001). The structure is consists of a 5-membered thiozolium ring joined to a six membered aminopyrim group. Its derivatives consist of side chains which differentiate it from their parent compound. Thiamine has a positive charge and also heat stable in acidic environments (Herman et al, 2011). Thiamine Pyrophosphate (TPP) is an active form of thiamine and it is a cofactor of several enzymes involved in carbohydrate metabolism. TPP is important for the production or storage of energy through glucose metabolism and the conversion of glucose from o fats which are then stored in adipose tissue. It is a cofactor of enzymes involved in the oxidative decarboxylation of compounds that are intermediates of the Krebs cycle. TPP is also associated with the alteration of the activities of neuronal and neuromuscle transmission to achieve a desired result (Preedy, 2013) The absorption of thiamine is accompanied by the phosphorylation of the vitamin. Thiamine treated in the lab is not consumed since most thiamine derivatives are absorbed more easily and at faster rates due to their high bioavailability. Some of these derivatives such as thiochrome contain toxins which have harmful effects on the production of energy in the cell and eventually lead to the death of an organism (Herman and Rima, 2011). Fluorometry is an analytical technique which involves the measurement of a radiant emission done by a compound as its makes an energy transition from a higher state to a lower state. The compound is brought into a state of higher energy by an excitation wavelength and an emission wavelength is measured as the molecule transits to a state of lower energy.(Wilson and Walker, 2011) This technique differs from spectrophotometry in that only the absorbed amount of radiation is measured in spectrophotometry (Sharma, 2007) METHOD AND MATERIALS

Extraction of thiamine from Cereal 6g of each cereal (Nestle’, Purity and ProNutro; RSA) was weighed out in duplicate. The samples were homogenized with 25ml 0.05M H2SO4 (MERCK, RSA) in 60ml centrifuge tubes. The tubes were centrifuged at 8500xg for 25 minutes. The supernatants were collected while some of it was left behind with the pellet. The pellet was then re-extracted with 15ml H2SO4 accompanied by centrifugation at 8500xg for another 25 minutes. The supernatants were combined and collected in a 100ml beakers labelled S1 and S2 for Sample 1 and Sample 2 respectively. Complex formation of thiamine from cereals and standard thiamine 25 ml of 2mg/100ml thiamine-HCl (Sigma-Aldrich, Germany) was prepared from diluting 10ml of 5mg/100ml thiamine-HCl. 10ml of the diluted thiamine-HCl was poured in duplicate in containers marked T1 and T2. These solutions were then treated in the same way as S1 and S2 for the remainder of the experiment. 10 ml of 15% NaOH (Rochelle Chemicals, RSA) was added to all the beakers. The solutions were stirred for 10 minutes. The pH of each solution was checked through paper indicators. 5ml ferricyanide (Saarchem, RSA) was added to all the beakers and they were stirred for a further 10 minutes at room temperature. 25ml of isobutanol (Rochelle Chemicals, RSA) was added to all the tubes and the solutions were stirred for another 10 minutes. The samples were centrifuged at 1400xg for 5 minutes. The supernatant of each tube was gently applied over 3g of anhydrous sodium sulphate (Rochelle chemical, RSA) in a glass funnel with a glass wool as stopper. Standard Curve for thiamine determination Samples for the standard curve were prepared in duplicate using T 1 and T2 as in table 1. Table 1: Sample preparation of thiamine-HCl samples for the standard curve to be used for [thiamine] determination Tube Volume thiamine-HCl (T1/T2) Volume pure isobutanol

1 0.2

2 0.3

3 0.5

4 1

5 2

6 3

4.8

4.7

4.5

4

3

2

RESULTS Table 2: Table showing the fluorescence values of thiamine-HCl from T1 Test Tube No. Isobutanol 1 2 3 4 5 6

Fluorescence reading 3.554 5.728 4.575 14.38 60.34 143.2 339.4

Thiamine Value 0 2.174 1.021 10.826 56.786 139.646 335.846

% F1 0% 0.65% 0.304% 3.22% 16.9% 41.58% 100%

Table 3: Table showing the fluorescence values of thiamine-HCl from T2 Test Tube No. Isobutanol

Fluorescence reading 3.542

Thiamine Value 0

% F2 0%

1 2 3 4 5 6

15.88 66.62 57.24 148.4 342.4 424.2

12.338 63.078 53.698 144.858 338.858 420.658

2.93% 15% 12.8% 34.4% 80.6% 100%

Table 4: Table showing the average fluorescence, standard deviation and the concentration of thiamineHCl from T1 and T2 Test Tube No.

%F1

%F2

Average %F

Isobutanol 1 2 3 4 5 6

0% 0.65% 0.304% 3.22% 16.9% 41.58% 100%

0% 2.93% 15% 12.8% 34.4% 80.6% 100%

0% 1.79% 7.65% 8.01% 25.65% 61.09% 100%

Standard deviation 0% 1.14% 7.35% 4.79% 8.75% 19.51% 0%

[Thiamine] ng/ml 0 32 48 80 160 320 480

Figure 1: Figure showing standard curve of the flourescenece of thiamine-HCL samples of known concetrations

120

100

Avg %Flourescene

f(x) = 0.22 x − 6.58 R² = 0.99 80

60

40

20

0

0

100

200

300

400

500

600

[Thiamine] ng/ml

Table 5: Table showing the thiamine content in the cereals that was determined from the standard curve in comparison to the thiamine concentration relayed on the cereal boxes Type of Cereal Caramel Flavor Purity Carrot

Sample No. Average S 1 S 2 S

34.41

22

28.21

24.86

23.71

24.29

7.212

7.028

7.12

Average of S1 and S2 26.25

[Thiamine] ng/ml

Dilution Factor

[Thiamine] ug/6g

[Thiamine] mg/100g

160.56

25X

4.014

0.0684

[Thiamine] from cereal box 0.8g/100g

10.16

89.37

25X

2.234

0.037

0.6g/100g

and Spinach Cerelac Regular Cerelac

ProNutro

Gluten free Purity Peach and Apricot Purity

1 S 2 S 1 S 2 S 1 S 2 S 1 S 2 S 1 S 2

13.29

13.11

13.20

5.644

5.67

5.66

4.878

5.068

4.98

5.257

5.301

5.28

5.445

5.340

5.39

45.52

46.36

45.94

40.43

39.88

40.16

14.45

14.58

14.52

14.79

14.75

14.77

5.32

67.95

25X

1.699

0.028

0.9g/100g

4.835

65.81

25X

1.645

0.027

0.6g/100g

43.05

234.90

25X

5.873

0.09

1.6g/100g

14.645

109.21

25X

2.730

0.045

1mg/100g

DISCUSSION Effect of the reagents Used Thiamine was partially hydrophobic which meant that it dissolved in water. It does not dissolve in organic solvents properly which meant that using butanol would ensure for separation during centrifugation in the extraction of thiamine (Song et al, 2008). It was also discovered that thiamine was unstable under basic conditions in the pH range of 8-10. NaOH was used to bring it to this pH and to provide hydroxyl molecule for nucleophilic attacks. At this pH an OH group could easily attack the second carbon of the ring structure which lead to a steady formation of a base-like intermediate and the opening of the ring. This process was facilitated by a redox reaction that was made possible by ferricyanide which acted as the oxidizing agent. The resulting structure was a thiamine derivative which expelled a water molecule to produce a yellow stable compound (Song et al, 2008). This compound was fluorescent and could be measured using a spectrofluorometer (Rocha et al, 2003) The sulfuric acid digested the thiamine in a similar manner that stomach acid does which also preserved the yellow stable compound in aqueous solutions and at various temperatures (Leichter and Joslyn, 1969) The difference of Thiamine concentration After the experiment the cerelac products were found to have the greatest yield, the proNutro had a greater yield and the purity had the least yield. This was due to the manufacturing processes that were used to produce each product. The presence of preservatives could have been one of the reasons for this. Purity was a cereal for babies that are months old which meant that all the nutrients need to be resistant to chemical and physical processes (Rapala-Kosik et al, 2009). This was because babies do not consume as much as adults do as such whenever babies consume food it has to count in terms of nutritional content. Uses this analogy it can be said that proNutro and cerelac had less preservatives

they were targeted to infants of about 1-5 years of age who had other vitamin sources. The preserving reagents that were used in the manufacturing process also play a role in the thiamine yield. For example if a sulfite was used as part preserving reagents, it would be more difficult to obtain the desired product as this reagent maintains the thiamine in one form over a wide range of physical and chemical conditions. Therefore, purity had more preservatives than the other cereals which lead to a small yield (Preedy, 2013). Errors The regular cerelac cereal samples that was analyzed only contained one extraction of thiamine, the second extraction was unwilling neglected, hence, the concentration of thiamine from the combined extracts could have yielded the maximum thiamine concentration that would be equal to that the one on the cereal box. CONCLUSION The purity cereals yielded the least amount of thiamine, the proNutro cereals yielded a larger thiamine concentration and the cerelac yielded the most thiamine concentration. This was a direct consequence of the preserving methods that were applied during the manufacturing processes of the cereals. REFERENCES Coiltate, T., (2001), Food: The Chemistry of its Components, Volume 25, Royal Society of Chemistry ProQuest ebrary, Cambridge, pp. 264-268 Elisa, K., Lemos, C., Azevedo, I & Martel, F., (2006). Characteristics of thiamine uptake by the BeWo Human Trophoblast Cell Line, Journal of Biochemistry and Molecular biology, 39 (4): 383-393 Herman, W. & Rima, O., (2011), Vitamins in the Prevention of Human Diseases, Walter du Gruyter ProQuest ebrary, Berlin, pp. 41-45 Leichter, J., and Joslyn, M.A., 1969. Kinetics of thiamin cleavage by sulphite, Biochemical Journal, 113: 611–615 Preedy, V.R., (2013), B Vitamins and Folate: Chemistry, Analysis, Function and Effect, The Royal Society of Chemistry, London, pp. 55-64 Rapala-Kozik, M., Golda, A., and Kujda, M., 2009. Enzymes that control the thiamine diphosphate pool in plant tissues. Properties of thiamine pyrophosphokinase and thiamine-(di)phosphate phosphatase purified from Zea mays seedlings, Plant Physiology and Biochemistry. 47: 237–242. Rocha, F.R.P., Filho. O.F., and Reis, B.F., 2003. A multicommuted flow system for sequential spectrophotometric determination of hydrosoluble vitamins in pharmaceutical preparations, Talanta, 59: 191–200 Sharma, D.C., (2007), Practical Medical Biochemistry, B.I. Publications, New Delhi, pp. 73 Song, J., Bettendorff, L., Tonelli, M., and Markley, J.L., 2008. Structural basis for the catalytic mechanism of mammalian 25-kDa thiamine triphosphatase, Journal of Biological Chemistry, 283: 10939–10948

Wilson, K. and Walker, J., (2011), Principles and Techniques of Biochemistry and Molecular Biology, 7th edition, Cambridge University Press, Cambridge, pp. 323...